Numb Inhibits Membrane Localization of Sanpodo, a Four-Pass Transmembrane Protein, to Promote Asymmetric Divisions in Drosophila  Kate M. O'Connor-Giles, James B. Skeath  Developmental Cell  Volume 5, Issue 2, Pages 231-243 (August 2003) DOI: 10.1016/S1534-5807(03)00226-0 Copyright © 2003 Cell Press Terms and Conditions

Figure 1 spdo Acts Upstream of the Intramembranous Cleavage of Notch to Regulate Asymmetric Divisions Dorsal and ventral views of wild-type (A and B) and spdo (E and F) nerve cords and, otherwise, wild-type (C and D) and spdo (G and H) nerve cords in which Notchintra was expressed throughout the CNS stained for Eve. Genotypes of embryos shown in (C) and (D) and in (G) and (H) are UAS-Nintra /+;pros-Gal4/spdoG104 and UAS-Nintra /+; pros-Gal4, spdoG104/spdoG104, respectively. (A and B) Each wild-type hemisegment contains one RP2 ([A], arrows), a cluster of five to six U neurons ([B], arrows), and a large cluster of Eve lateral (EL) neurons. The arrowhead in (A) points to an RP2sib that still has residual Eve expression. (C and D) Expression of Notchintra throughout the CNS results in a loss of RP2 neurons ([C], arrows), an increase in U neurons ([D], arrows), and a decrease in EL neurons ([D], arrowheads). (E and F) Each hemisegment in a spdo embryo contains two RP2s ([E], arrows), no U neurons ([F], arrows), and normal numbers of ELs ([F], arrowheads). (G and H) Notchintra expression throughout the CNS of spdo embryos results in a loss of RP2 neurons, an increase in U neurons ([H], arrows), and a decrease in EL neurons ([H], arrowheads). In (G), black arrows mark hemisegments with no RP2s; white arrow marks hemisegment with one RP2. The asterisk in H marks two RP2sibs with residual Eve expression. In all panels, anterior is up. (I) Table showing the transformation percentage of B daughter cells into A daughter cells upon generalized expression of Notchintra or NotchECN in the CNS of wild-type and spdo mutant embryos for the indicated sibling neurons. Developmental Cell 2003 5, 231-243DOI: (10.1016/S1534-5807(03)00226-0) Copyright © 2003 Cell Press Terms and Conditions

Figure 2 Molecular Identification of spdo (A) Schematic of genomic region between claret (ca) and brevis (bv) with relevant SNPs indicated. SNPs B and C define the spdo genomic region, which contains nine genes. (B and C) The CG31020 transcript is detected in the CNS, PNS, and mesoderm of wild-type, but not homozygous spdoZZ27 embryos. (D) Amino acid alignment and predicted topology of Drosophila and Anopheles Spdo. Red triangles, nonsense mutations; green triangles, missense mutations; purple brackets, internal deletion; blue boxes, predicted transmembrane domains; gray shading, 60-amino acid conserved region. (E–H) Stage 15 nerve cords of indicated genotype stained for Eve. (E) Wild-type hemisegments contain one RP2 neuron (large arrows) and five to six U neurons (arrows). (F) spdo mutant hemisegments contain two RP2s (large arrows) and no U neurons. (G) CG31020 RNAi-treated wild-type embryo exhibits an Eve CNS phenotype identical to that of spdo (compare to [F]). (H) spdo embryo in which CG31020 was expressed in the CNS exhibits a wild-type Eve CNS pattern (compare to [E]); large arrows mark RP2, and small arrows mark the U neurons. In (H), the genotype of the embryo is sca-Gal4/UAS-spdo; spdoG104. Anterior is left in (B) and (C) and up in (E)–(H). Developmental Cell 2003 5, 231-243DOI: (10.1016/S1534-5807(03)00226-0) Copyright © 2003 Cell Press Terms and Conditions

Figure 3 Spdo Appears to Be Expressed in All Embryonic Cells Known to Undergo Asymmetric Divisions Stage 11 (A–C and F–I), stage 10 (D and E), and stage 13 (J) wild-type (A, B, and D–J) and spdoZ143 (C) embryos labeled for Spdo (green), Hunchback ([E], red), Prospero ([G], red), Svp-lacZ ([H], red), Eve ([I], red), and Cut ([J], red). (A) Spdo protein localizes to the apical and basal side of the cell membrane of NBs and GMCs (the arrow marks the NB layer, and the arrowhead marks GMC layer). (B) Spdo protein localizes uniformly around the medial and lateral extent of NBs and to small- and intermediate-sized cytoplasmic puncta in most expressing cells (A, B, and D–J). (C) Spdo exhibits largely cytoplasmic localization in spdoZ143 embryos. (D–G) All NBs ([E], red) and all GMCs ([G], red) express Spdo. (H–J) Spdo is also expressed in Svp-lacZ ([H], red) and Eve ([I], red)-positive mesodermal cells and in all Cut-positive PNS cells ([J], red). Anterior is up in (B)–(G) and left in (A) and (H)–(J); apical is up in (A); scale bar, 20 μm. Developmental Cell 2003 5, 231-243DOI: (10.1016/S1534-5807(03)00226-0) Copyright © 2003 Cell Press Terms and Conditions

Figure 4 Spdo Colocalizes with Notch and Delta High-magnification views of stage 9 (A–C) and stage 11 (D–I) embryos labeled for Spdo (red) and Notch ([A–F], green) or Delta ([G–I], green). (A–C) In early NBs Notch (A and C) and Spdo (B and C) colocalize in large puncta near the cell membrane. (D–F) In GMCs, Notch (D and F) and Spdo (E and F) colocalize in smaller puncta near the cell membrane (arrows) and more diffusely throughout the cytoplasm (arrowheads). (G–I) In the GMC layer Delta-positive puncta (G and I) at the membrane reside in tight apposition to (arrows), or colocalize with (arrowhead), Spdo. Scale bars, 10 um; scale bar in (D) applies for (D)–(I). Anterior is up in all panels. Developmental Cell 2003 5, 231-243DOI: (10.1016/S1534-5807(03)00226-0) Copyright © 2003 Cell Press Terms and Conditions

Figure 5 Spdo Physically Associates with Notch and Numb In Vivo (A) Antibodies specific to the intracellular and extracellular domain of Notch immunoprecipitate Spdo. In control experiments mouse anti-Myc antibodies do not coprecipitate Spdo and neither Notch antibody coprecipitates EGFR. (B) Numb-specific antisera, but not preimmune sera, coimmunoprecipitates Spdo, but not EGFR. (C) Antibodies specific for EGFR do not immunoprecipitate Spdo. In each panel, lane 1 contains precleared embryonic lysate equal to one-tenth of the input for the immunoprecipitation assays. Developmental Cell 2003 5, 231-243DOI: (10.1016/S1534-5807(03)00226-0) Copyright © 2003 Cell Press Terms and Conditions

Figure 6 Numb Inhibits the Membrane Localization of Spdo (A–C) Lateral section of the CNS of a stage 11 wild-type embryo. Cells with low levels of Numb (red) generally localize Spdo (green) to the cell membrane (arrowhead). Cells with high levels of Numb generally localize Spdo to the cytoplasm (arrow). The asterisk marks an NB with high-level Numb and largely cytoplasmic Spdo. (D and E) Ventral sections of the CNS in late stage 12 wild-type and numb2 embryos. In wild-type, Spdo accumulates in puncta near the cell membrane and in the cytoplasm (D). In numb2 embryos, Spdo accumulates at high levels around the entire cell membrane of most cells and exhibits reduced cytoplasmic accumulation (E). (D) and (E) were obtained from identically staged wild-type and numb embryos from the same staining reaction with identical parameters. (F) Schematic of d/vMP2 sibling neurons and the focal planes of the images shown in (G)–(J). (G–J) Sections showing newly born dMP2 or vMP2 neurons in stage 11 wild-type or numb2 embryos. In wild-type, Spdo (green) localizes to the cell membrane of vMP2 ([G], red) and to cytoplasmic puncta in dMP2 ([H], red). (I and J) In numb embryos, Spdo (green) localizes to the membrane of vMP2 ([I], red) and dMP2 ([J], red) and exhibits minimal cytoplasmic accumulation in either cell. The scale bars are 10 μm in (A)–(E) and 5 μm in (G) and (H). Anterior is left and apical in (A)–(C) and up in (D)–(J). Developmental Cell 2003 5, 231-243DOI: (10.1016/S1534-5807(03)00226-0) Copyright © 2003 Cell Press Terms and Conditions

Figure 7 Model of spdo/Notch/numb-Dependent Asymmetric Divisions In the A cell Spdo localizes to the cell membrane, where it promotes active Notch signaling. In the B cell, the presence of Numb inhibits the cell membrane localization of Spdo. In the absence of Spdo protein at the cell membrane, productive Notch signaling does not occur in the B cell (see text for details). Developmental Cell 2003 5, 231-243DOI: (10.1016/S1534-5807(03)00226-0) Copyright © 2003 Cell Press Terms and Conditions